There is a mounting concern that terrorist groups may possess or can obtain their own stocks of variola virus, the etiological agent of smallpox infection. To respond to this specific threat, the United States must have at its disposal supplies of both vaccinia virus vaccine and antiviral compounds directed against smallpox infection. The antiviral compounds are needed in situations in which the vaccine is contraindicated, such as immunosuppression. In addition, the antiviral compounds are needed to blunt adverse complications that are known to be associated with vaccine administration. An ideal antiviral should also be able to directly prevent smallpox infection in situations where vaccine delivery is delayed. Toward this end, antivirals directed against the vaccinia virus DNA polymerase (V AC-POL) and processivity factor (V AC-PF) have great potential. In general, PFs act as clamps that slide along the DNA and bind to their respective POLs. Through this POL/PF interaction, DNA synthesis will be processive, generating new DNA strands that are thousands of nucleotides long. Viruses that have mutated PFs are defective in replication since POL alone can synthesize only short DNA products (as few as three nucleotides). A strongly favorable feature of PFs is that they function with their own POLs, so that an antiviral that targets a viral PF should be very specific and not interfere with cellular replication. V AC-POL and V AC-PF interaction has been recently characterized and both proteins can be purified and combined to function in processive DNA synthesis. The goal of this project is to discover antivirals that will specifically block processive DNA synthesis by V AC-POL and V AC-PF. A newly developed mechanistic rapid plate assay will be used to screen thousands of potentially inhibitory compounds. This high throughput screening will be followed by procedures to evaluate the inhibitory compounds and to ultimately test their ability to block vaccinia virus infection. A drug that prevents vaccinia processive DNA synthesis will be useful in curtailing vaccinia vaccine complications. The same drug may also directly block variola infection since the POLs and PFs, respectively, of vaccinia and variola have 97% direct homology. The approach may help deliver the 'just-in-time' need for reagents to combat a smallpox bioterrorism threat.